December 18, 2020

Challenges limit bioproduction

Secretion challenges: ER Trafficking

Using data science for rational bioproduction

Quantifying demands

Ribo-Seq methods for quantification

Distinguishing translation events on the ER

Demands in Komagataella phaffii

Gene targets for enchanced expression

Cotranslationally Targeted Proteins
Gene cTPM Length Description
PST1 2374 418 Cell wall protein
PMA1 1968 896 Plasma membrane H+-ATPase
KAR2 1325 678 Protein import-associated ATPase
GAS1 772 538 1,3-beta-glucanosyltransferase (isoform 1)
ERG5 314 528 C-22 sterol desaturase
Gene cRPM Length Description
PMA1 4772 896 Plasma membrane H+-ATPase
PST1 3499 418 Cell wall protein
KAR2 2502 678 Protein import-associated ATPase
GAS1 1324 538 1,3-beta-glucanosyltransferase (isoform 1)
SIT1 568 633 Ferrioxamine B transporter (isoform 1)
Posttranslationally Targeted Proteins
Gene cTPM Length Description
SCV12161.1 17336 55 Uncharacterized protein
SPI1 10900 135 GPI-anchored cell wall protein
CCA36283.1 10863 93 Uncharacterized protein
XP_002494332.1 10762 65 Uncharacterized protein
YNL190W 1856 132 Uncharacterized protein
Gene cRPM Length Description
SPI1 6140 135 GPI-anchored cell wall protein
CCA36283.1 4158 93 Uncharacterized protein
SCV12161.1 3603 55 Uncharacterized protein
XP_002494332.1 2619 65 Uncharacterized protein
YNL190W 1005 132 Uncharacterized protein

PMA1

Description
PMA1 is a plasma membrane H ATPase that acts as the primary regulator of cytoplasmic pH.

  • Essential gene

  • Long lived

Demands
+ Represents 10% of nascent chains entering ER cotranslationally

SPI1

Description
SPI1 is a GPI-anchored cell wall protein involved in weak acid resistance

  • Non-essential gene

  • Weakened chemical and acid pH resistance

Demands

  • Represents 18% of nascent chains entering ER posttranslationally

Testing under heterologous conditions

Changes in demands (skepticle)

Highest Differences in Expression for Cotranslationally Targeted Proteins
Gene Before After Description
AOA66821.1 31 45512 Hypothetical protein
AOA65731.1 496 13749 Hypothetical protein
FMP45 92 10023 Integral membrane protein localized to mitochondria
AOA70177.1 264 9564 Hypothetical protein PAS_chr4_0947
OLE1 1678 10698 Delta(9) fatty acid desaturase
HSP150 4522 12420 O-mannosylated heat shock protein
NCE102 7129 13812 Protein of unknown function
BXI1 369 4241 Protein involved in apoptosis
CTR1 989 4649 High-affinity copper transporter of the plasma membrane
YDR262W 276 3024 Putative protein of unknown function
MSC1 16 2674 Protein of unknown function
DIP5 1234 3700 Dicarboxylic amino acid permease
YDR134C 279 2480 Cell wall protein
ITR1 299 2238 Myo-inositol transporter
GAL2 1698 3468 Galactose permease
Highest Differences in Expression for Postranslationally Targeted Proteins
Gene Before After Description
TIP1 51 60146 Major cell wall mannoprotein with possible lipase activity
AOA67300.1 13 15467 Hypothetical protein
SCV12161.1 51308 63838 Hypothetical protein
PRB1 192 4712 Vacuolar proteinase B (yscB) with H3 N-terminal endopeptidase activity
MKC7 53 1575 GPI-anchored aspartyl protease
AOA67261.1 3323 3910 Hypothetical protein
NPC2 1383 1724 Functional homolog of human NPC2/He1
AOA70251.1 3 117 Hypothetical protein PAS_chr4_0359
FLO1 5 89 Lectin-like protein involved in flocculation
AOA66439.1 59 130 Hypothetical protein
LCL2 310 372 Putative protein of unknown function
WSC2 25 59 Sensor-transducer of the stress-activated PKC1-MPK1 signaling pathway
AOA70184.1 3 37 Hypothetical protein PAS_chr3_0002
YNL190W 13 44 Hydrophilin essential in desiccation-rehydration process
NPR2 17 35 Subunit of SEA (Seh1-associated), Npr2/3, and Iml1p complexes